The membrane-bound transpeptidase which catalyzes peptide crosslinking during wall peptidoglycan synthesis in bacteria is the lethal target of penicillin (i.e. penicillins and cephalosporins). Exocellular transpeptidases excreted by Streptomyces R61 and R39, respectively, were purified to protein homogeneity. They may be soluble forms of the corresponding membrane-bound enzymes. Membrane-bound transpeptidases of streptomyces R61, K15 and rimosus, and of streptoccus faecalis and Escherichia coli K12 were solubilized and some of them were partially purified. Exocellular and membrane-bound enzymes catalyze: 1) transfer reactions involving donor and accceptor peptides, 2) hydrolysis of donor peptides in the absence of acceptor and 3) breakdown of penicillin into a biologically inactive compound. The objectives are: 1) final purification of the various solubilized membrane transpeptidases, 2) relationship between the exocellular and membrane-bound transpeptidases of Streptomyces R61, 3) relationship between the exocellular transpeptidase ad penicillinase of streptomyces R39 (which excretes both enzymes), 4) identification of the chemical modifications undergone by the antibiotic during its interaction with the exocellular and solubilized membrane transpeptidases, 5) identification of the enzyme centers involved in hydrolysis, in transfer reactions and in interaction with the antibiotic and 6) identification and quantitation of the parameters which lead to intrinsic sensitivity or resistance of the transpeptidases to penicillin.